INFLUENCE OF CA2+ ON CONFORMATION AND STABILITY OF 3 BACTERIAL HYBRIDGLUCANASES

The three hybrid glucanases (1-12)AMY . MAC(13-214), (1-12)AMY . des-T yr13MAC(14-214); (1-16)AMY . MAC(17-214) are composed of short N-termi nal segments of 12 or 16 amino acid residues derived from the Bacillus amyloliquefaciens glucanase (AMY) and of residues 13-214, 14-214 and 17-214, respectively, derived from the Bacillus macerans enzyme (MAC). The three proteins have similar conformational features as shown by t he similar characteristics of their CD spectra in the far- and near-ul traviolet region. A metal-ion-binding site was identified in the hybri d glucanase (1-16)AMY . MAC(17-214) by a crystal structure analysis [K eitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Ac ad. Sci. USA 90, 5287-5291]. Only minor conformational changes of the three hybrid glucanases were observed depending on the presence or abs ence of Ca2+ ions but for (1-16)AMY . MAC(17-214) and (1-12)AMY . des- Tyr13MAC(14-214) the occupation of this metal-binding site by a Ca2+ i on is connected with a large increase of the stability against thermal and chemical unfolding. Surprisingly, for (1-12)AMY . MAC(13-214), wh ich differs from (1-12)AMY . des-Tyr13MAC(14-214) by only one addition al amino acid in an N-terminal loop region, the effect of Ca2+ ions on the stability is small. The exchange of a few amino acid residues nea r the N-terminus of the B. macerans glucanase against amino acids foun d at comparable positions in the B. amyloliquefaciens glucanase seems to influence very strongly the strength of the Ca2+ binding site and c oncomitantly the stability of the hybrid glucanases.